I'm reading a 1979 paper about GTA (Yen, Hu and Marrs, Journal of Molecular Biology 131:157-168). The authors devised a way to select for mutants that overproduced GTA, and used these to find out more about how the particles are produced and the DNA they contain.

The mutant they focused on produces enough GTA that culture supernatants transfers any one chromosomal marker to about 0.0001 to 0.001 of the cells in a recipient culture. As each particle contains about 0.001 of the donor chromosome, and recombination of such short fragments is relatively inefficient, this means that the supernatant probably contains about as many particles as there are cells in the recipient culture. That was probably about 10^9 per ml.

The mutant grew poorly, and the authors interpreted this as a consequence of increased cell lysis associated with the increased GTA production. They maintained the strain by growing it in medium they had found to inhibit GTA production (PYE medium), and transfered it to a GTA-inducing medium (RCV) when they wanted GTA. After this transfer they observed that after several cell divisions about 10-20% of the cells died at the same time that high levels of GTA became detectable in the medium.

This is a very provocative result (too bad they don't show any data), because it implies that GTA production is very deleterious . I'd never heard of either medium; I wanted to find out what's in them but one of the disadvantages of reading old papers is that they and their references are often not available online. But simply Googling "PYE RCV" led me to the recipes - apparently they're quite widely used. PYE is just 0.3% peptone and 0.3% yeast extract, which makes it a slightly more dilute version of our old favourite rich medium LB. RCV is a defined medium used for R. capsulatus photosynthetic growth; it contains 0.4% malic acid as the only carbon source, 0.1% ammonium sulfate, thiamine, and other salts specified in papers that Springer will show me for $32.

So cells make lots of GTA in rich medium but not in the very poor medium used for photosynthetic growth. Hmmm...

But searching for the paper with the recipes for these media led me to an even older paper that I need to first read. This is Marrs 1974, PNAS 71:971-973, and PNAS is on line all the way back to the beginning. So I'll take a break to read this paper, and post on it before continuing.

Yesterday I found time to sit down with my colleague who works on the 'gene transfer agent' (GTA) of Rhodobacter capsulatus. This helped me sort out a few things that are known about this entity, and a few things that aren't.

Is GTA derived from phage? Almost certainly. My colleague's lab's recent work has shown that some of the genes needed for GTA production are homologs of known phage genes. Old work from Barry Mrrs' group also showed that supernatants of GTA-producing cultures contain particles that look like tiny tailed phages. However, no GTA- control cultures were examined, and these phage-like particles could be produced from a defective prophage unrelated to GTA.

GTA particles contain chromosomal DNA fragments about 4.5kb, but nothing is known about how the DNA comes to be packaged in these particles. This information is critical to understanding how evolutionary processes act on GTA.

Old Cot-curve and restriction analyses were consistent with the fragments being derived from random positions in the chromosome, but the resolution is very poor. The issue could be nicely resolved by isolating DNA from the particles and hybridizing it to Affymetrix chips. Unfortunately my colleague says that getting sufficient GTA particles is quite difficult, as yields are both very low and not very predictable. An attempt to find out whether the ends of the fragments are blunt or staggered was unsuccessful.

From an evolutionary perspective, the most critical missing pieces of information are probably whether GTA is always (or often) accompanied by the death of the producing cell, and whether genes allowing GTA production can be transferred by GTA. That's because, if the genes are anything more than accidents of evolutionary history, they must either enhance the fitness of the cells they are in or spread into new cells faster than they kill their present cells.

If cells can produce GTA without dying, they must have a way to pass the particles out through the cell membranes without destroying them. Some filamentous phages can be secreted by living cells, but I think the tailed phages GTA is thought to resemble escape only by lysing their hosts. The amounts of GTA produced are sufficiently small that this might entail death of only a tiny fraction of the culture.

And if GTA does kill its cells on the way out, GTA could persist of evolutionary time only if it either spread between cells like an infectious agent or greatly increased the fitness of its close kin. Neither of these seem very likely, but I'll post more about this later.

The post-docs have finished the first-pass analysis of how E. coli gene expression is affected by both the E. coli Sxy and the H. influenzae Sxy proteins. I suppose I shouldn't be surprised that it's more complicated than I had hoped. For example, unlike the situation in H. influenzae, in E. coli there are also groups of genes whose expression goes down when Sxy is present.

One complication is that these cells are probably seriously OVER-producing Sxy. Unlike H. influenzae, where we've only done arrays of cells expressing a single-copy sxy gene under its natural promoter, these E. coli studies used a sxy gene on a high-copy plasmid and under a highly inducible promoter. We know that prolonged expression of Sxy from this plasmid produces large quantities of denatured Sxy (in inclusion bodies) and we don't know the extent to which even the 30-minute expression used for the array studies might create a situation unlike that of natural sxy expression.

This isn't a real research post, but my friends/colleagues thought I was wrong when I explained this to them so I want to see what others think.

I live in a condo and my apartment is heated by electric baseboard units (call this heating electricity). Like everyone else I also use electricity to accomplish domestic tasks such as lighting, cooking and refrigeration (call this working electricity).

I argue that the inefficiency with which I use working-electricity processes in my home is irrelevant to my electricity consumption because all the electricity used by these processes ultimately becomes heat. This applies not only to 'wasted' energy such as the heat put out by light bulbs, but to the work I'm using the electricity for, such as the light itself. That's because work becomes heat; for example light becomes heat when it is absorbed by the surfaces it hits. Thus every watt of electricity I use for cooking or lighting sooner or later becomes heat, and as such proportionately reduces the amount of electricity I need to send directly to the heaters. In effect I'm getting my working electricity for free.

The argument doesn't apply to working energy that gets lost as light out the windows or sound through the walls or hot water down the drain. And maybe not to the energy equivalent of the information I'm transmitting to Blogger with this post, though I suspect that is somewhere between infinitesimal and nonexistent. But it applies to everything that happens in the apartment.

The argument also doesn't apply when the weather is warm enough that any heating needed is less than the heat produced by working electricity. And if the weather ever got hot enough that I used electricity for cooling, I'd be paying double for the energy wasted by my working electricity - e.g. once to run the computer and once to run the air conditioner to get rid of the heat.

And it wouldn't completely apply in winter if I was able to use natural gas or a similarly cheap energy source for heating. But, given that I'm stuck with expensive electrical heating, I console myself with the thought that all the rest of my electricity is free.

A colleague's lab has been working on the molecular biology of the 'Gene Transfer Agent' (GTA) of the bacterium Rhodobacter capsulatus. He and I have very different ideas about the evolutionary function of GTA, and we plan to sit down together and work through our disagreements, maybe coming up with a synthesis as a review article. I haven't been paying close attention to GTA, and in this post I'm going to take the first step by summarizing what I think I remember about it (before I go back and read any papers).

The phenomenon: Cultures of R. capsulatus have been known for many years to produce small phage-like particles, each consisting of a protein coat surrounding a 3-4kb fragment of R. capsulatus DNA. These particles can be separated from the source culture and are able to introduce their DNA into other R. capsulatus cells, where it can recombine with the chromosome and change the recipient cell's genotype. The variety of genes that can be transferred suggests that the DNA fragments may be random segments of the source cell's DNA.

I read about GTA when I was in grad school in the 1980s and first becoming interested in the evolution of processes causing gene transfer. I was already coming to the heretical conclusion that bacterial gene transfer by conjugation and transduction occurs as accidental side effects of infectious processes, not because such transfer is beneficial to the cell. At that time only Barry Marrs' lab had worked on GTA. My supervisor, the phage biologist Allan Campbell, thought that GTA was probably produced by a defective prophage. I was working on a cryptic prophage at the time, and this made sense to me. GTA would then be a form of transduction, a side effect of activity of genes whose normal function is to package phage DNA so it can infect new host cells.

The genes: More recently my colleague's lab has identified the R. capsulatus genes responsible for production of GTA and has partially characterized their regulation. As I recall, these genes are in a couple of clusters that do resemble defective prophage but that also have some properties of normal genes. In particular, aspects of the regulation suggest selection for a cellular function. My colleague has also done some analysis of the distribution of the GTA-producing genes, and as I recall this was not consistent with a single acquisition of a defective prophage. He thus interprets his findings as evidence that the ability to transfer genes by GTA is beneficial to R. capsulatus, i.e. that GTA has evolved as a form of bacterial sex.

Questions that I think have not yet been answered, or whose answers I forget: Does the individual cell that produces GTA die, as phage-infected cells normally do? Do only a small fraction of cells in a R. capsulatus culture produce GTA? How many genes are specific to GTA production (have no other function in the cell)? Have phage-derived genes acquired cellular functions independent of GTA production? Does GTA production directly reduce fitness? Can the ability to produce GTA be transferred by GTA? How strong is the phylogenetic evidence?

Next steps: Perhaps we should start our collaboration by working our way through the GTA literature, starting with Barry Marrs' 1974 PNAS paper. This would have the advantage of giving us both the same foundation of facts and factoids (things that look like facts but later turn out to be wrong) to base our discussions on. At the same time we ought to read one or more papers that clarify the evolutionary issues. My "Do bacteria have sex" paper is an obvious choice but shouldn't be the only one.

I'll ask my colleague to read this post, and we can then set up a time for our first meeting and decide what we should read in preparation for it.